Fig 1.
Overview of metabolites labelled from D-glucose in bloodstream form T. brucei.
Metabolic network derived from detected metabolites and annotated enzymes in T. brucei. Red nodes indicate labelled metabolites from 50% U-13C-glucose, with darker red indicating higher percentage labelling. Grey nodes indicate no labelling detected. Nodes without spots indicate that the metabolite was not detected, but the presence is suggested by adjacent metabolites in the network.
Fig 2.
Labelling of glycolytic intermediates from 50% U-13C-glucose in BSF T. brucei.
(A) Schematic of the glycolytic pathway in BSF T. brucei. Black font = detected on LCMS, grey font = inferred intermediates not detected in this assay. (B) Heatmap of relative isotopologue abundances for glycolytic intermediates after labelling for 24 hours with 50% U-13C-glucose in HMI11. 0 = all carbons unlabelled (e.g. U-12C-glucose), 6 = six carbons labelled (e.g. U-13C-glucose). (C) Isotopologue abundances of hexose phosphates after labelling for 24 hours with 50% U-13C-glucose (n = 3, mean ± SD). (D) Isotopologue abundances of glucose 6-phosphate after labelling for 24 hours with 100% U-12C-glucose (white columns), 50% U-13C-glucose (hatched columns) or 100% U-13C-glucose (black columns) (n = 3, mean ± SD). Abbreviations: G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GA3P, glyceraldehyde 3-phosphate; 1,3BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; G3P, glycerol 3-phosphate. Enzymes: 1, hexokinase; 2, glucose-6-phosphate isomerase; 3, phosphofructokinase; 4, fructose-1,6-bisphosphatase; 5, aldolase; 6, triosephosphate isomerase; 7, glyceraldehyde 3-phosphate dehydrogenase; 8, phosphoglycerate kinase; 9, phosphoglycerate mutase; 10, enolase; 11, pyruvate kinase; 12, alanine aminotransferase; 13, glycerol-3-phosphate dehydrogenase; 14, glycerol kinase; 15, mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase; 16, methylglyoxal detoxification pathway.
Fig 3.
Labelling of pentose phosphate pathway and purine nucleotides from 50% U-13C-glucose in BSF T. brucei.
(A) PPP in BSF T. brucei. Black font = detected on LCMS, grey font = inferred intermediates not detected in this assay. (B) Heatmap of relative isotopologue abundances for pentose phosphate pathway intermediates after labelling for 24 hours with 50% U-13C-glucose in HMI11. (C) Representative mass spectra for octulose 8-phosphate from T. brucei incubated with unlabelled (U-12C) glucose (top) and labelled (U-13C) glucose (bottom). The +3, +5 and +8 isotopologues are predominant in the labelled sample. (D) Chromatograms for all octulose 8-phosphate isotopologues from T. brucei incubated with unlabelled (U-12C) glucose, labelled (U-13C) glucose, unlabelled (U-12C) ribose and labelled (U-13C) ribose. (n = 3). (E) Heatmap of relative isotopologue abundances for purine nucleotides and cofactors after labelling for 24 hours with 50% U-13C-glucose in HMI11. Abbreviations: G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; 6PG, 6-phosphogluconate; Ru5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; O8P, octulose 8-phosphate; N9P, nonulose 9-phosphate; GA3P, glyceraldehyde 3-phosphate. (F) The T. brucei transaldolase gene was heterologously expressed in E. coli and the protein purified (left hand panel, with the crude E. coli extract on the left and purified protein to the right). Purified transaldolase was shown to produce octulose 8-phosphate when provided with ribose 5-phosphate and fructose 6-phosphate as acceptor and donor substrates respectively (right hand panel). Enzymes: 1, Hexokinase; 2, glucose 6-phosphate isomerase; 17, glucose 6-phosphate dehydrogenase; 18, 6-phosphogluconate dehydrogenase; 19, ribose 5-phosphate isomerase; 20, ribokinase; 21, transaldolase.
Fig 4.
Glucose-derived dicarboxylic acid and pyrimidine metabolic pathways in BSF T. brucei indicate activity of PEPCK.
(A) Metabolic pathways of dicarboxylic acid and pyrimidine synthesis in BSF T. brucei. Red carbons in structures indicate the predominant 13C labelling. (B) Heatmap of relative isotopologue abundances for dicarboxylic acid and pyrimidine intermediates after labelling for 24 hours with 50% U-13C-glucose in HMI11. (C) Isotopologue abundances of dTTP after labelling for 0 (U), 1 (L1) and 24 (L24) hours with 50% U-13C-glucose in HMI11 (H) or CMM (M) (n = 2–3, mean ± SD). Extensive de novo dTTP synthesis was only observed in CMM (i.e. in the absence of exogenous thymidine). Abbreviation: PEP, phosphoenolpyruvate; Pyr, pyruvate; OxAc, oxaloacetate; Mal, malate; Fum, fumarate; Succ, succinate; Asp, Aspartate; AdSucc, adenylosuccinate; DHO, dihydroorotate; Oro, orotate; Ura, uracil; Urd, uridine; dUrd, deoxyuridine; Tmd, thymidine. Enzymes: 11, pyruvate kinase; 22, malate dehydrogenase; 23, fumarase; 24, NADH-dependent fumarate reductase); 25, aspartate aminotransferase; 26, adenylosuccinate synthase; 27, adenylosuccinate lyase; 28, aspartate carbamoyltransferase and dihydroorotase; 29, dihydroorotate dehydrogenase; 30, orotate phosphoribosyltransferase and orotidine 5-phosphate decarboxylase; 31, nucleoside diphosphatase; 32, ribonucleoside-diphosphate reductase; 33, thymidylate kinase; 34, thymidylate synthase; 35, thymidine kinase; 36, uracil phosphoribosyltransferase; 37, uridine phosphorylase; 38, nucleoside diphosphate kinase; 39, cytidine triphosphate synthase.
Fig 5.
PEPCK is essential to bloodstream form T. brucei.
(A) Growth curve of two different clones showing down-regulation of pepck by RNAi (RNAiPEPCK-D6 and RNAiPEPCK-B3) and the parental 427 BSF strain (WT) incubated in the presence (.i) or in the absence (.ni) of 10 μg/ml tetracycline. Cells were maintained in the exponential growth phase (between 105 and 2x106 cells/ml) and cumulative cell numbers have been normalized for dilution during cultivation. (B) Western blot analyses of the parental (WT) and mutant cell lines with the anti-PEPCK and anti-enolase sera containing antibodies as indicated in the left margin. (C) Western blot demonstrates significantly higher levels of PEPCK in procyclic form (PF) compared to bloodstream form (BF) WT T. brucei. *Analyses were performed on 5 x 106 cells per sample and HSP-60 is shown as the loading control.
Fig 6.
Metabolic profile of RNAiPEPCK-B3 BSF T. brucei confirms depletion of dicarboxylic acids and pyrimidines.
(A) Proton (1H) NMR analysis of excreted pyruvate and succinate from glucose. Pyruvate (Pyr) and succinate (Suc) excreted by the bloodstream form 427 cell line (WT), and the RNAiPEPCK-B3 mutant non-induced (ni), tetracycline-induced 2 days (i 2 days), and tetracycline-induced 5 days (i 5 days) was determined by 1H-NMR. The cells were incubated in PBS containing 4 mM glucose for 4 hours. Each spectrum corresponds to one representative experiment from a set of at least 3. A part of each spectrum ranging from 2.2 ppm to 2.4 ppm is shown. (B) LC-MS analysis of intracellular glycolytic intermediates. Relative isotopologue abundances following incubation in PBS containing 4 mM U-13C-glucose for 4 hours for metabolic intermediates from RNAiPEPCK-B3 mutant non-induced (NI) and tetracycline-induced for 2 days (I). Significant decreases in labelling were observed for PEPCK-derived metabolites including 3-carbon labelling in dicarboxylic acids and aspartate, and 2-carbon labelling in pyrimidine nucleotides (or 7-carbon labelling for ribose-containing metabolites). Labelling was not significantly decreased in glycolytic intermediates or in other glucose-derived metabolites, including alanine, acetate (shown here as acetyllysine) and ribose (shown here as 5-carbon labelling in UTP).
Table 1.
Production of succinate, pyruvate, alanine and acetate from glucose in PEPCK RNAi T. brucei measured by 1H-NMR.
Fig 7.
Glucose is a carbon source for acetate, lipids and sugar nucleotides in WT BSF T. brucei.
(A) Time-dependent incorporation of glucose into diverse pathways. Relative isotopologue abundances for unlabelled cells (0) and cells incubated with 50% U-13C-glucose in HMI11 for one hour (1) and 24 hours (24). Labelling reaches equilibrium within 1 hour for pyruvate, alanine, malate, aspartate and ribose 5-phosphate. Minimal labelling is observed in 2-hydroxyethyl-TPP after 1 hour, but complete (50%) labelling occurs within 24 hours, indicating a relatively slow flux towards acetate production by this pathway. (n = 3, mean ± SD). (B) Heatmap of relative isotopologue abundances for lipids after labelling for 24 hours with 50% U-13C-glucose in HMI11 indicating a combination of salvage mechanisms with de novo synthesized glycerol phosphate and limited incorporation of glucose-derived acetyl-CoA. After manual curation, no isotopologues containing more than 6 labelled carbons could be confirmed for the detected lipids. (C) Isotopologue abundances of UDP-N-acetyl glucosamine (UDP-GlcNAc) after labelling for 24 hours with 50% U-13C-glucose in HMI11 reveal incorporation of labelled carbon through glucosamine, acetate, ribose and uracil. Structures represent the likely isotopologue(s) for each mass with 13C atoms and bonds shown in red. Additional minor isotopologues have been omitted for clarity, the most abundant of which are isotopologues containing 3-labelled glucosamine 6-phosphate.